This invention relates to a support cell for processing samples, particularly biological samples, supported on solid supports. In addition the invention relates to automated apparatus for processing samples supported on solid supports, which automated apparatus may be used with the support cell of the invention. The invention also relates to a method of processing a sample supported on a solid support.
It is common practice, in certain technical fields, for samples of interest to be subjected to a particular process to be supported on solid supports. For example, protein or nucleic acid samples may be supported on filters or membranes: typically such filters or membranes are made of nylon or nitrocellulose. Samples, especially biological samples (e.g. cell suspensions or tissue sections) may also be supported on slides. Typically the slides are of transparent glass.
Samples supported on slides may be processed by a number of various techniques known to those skilled in the art, which include: standard staining techniques (e.g. Gram staining of micro-organisms, haemotoxylin/eosin staining of tissue sections); immuno- and enzyme cytochemistry (e.g. peroxidase/anti-peroxidase staining); ligand binding studies; in situ hybridisation of labelled DNA or RNA probes: and in situ amplification of nucleic acids present in a sample by means of polymerase chain reaction (PCR).
Typically, such processing is at present performed manually by laboratory personnel. This renders the processing slow and subject to individual variation (from one sample to the next, and/or from one day to the next). and is tedious for the personnel concerned. Some attempts have been made to provide apparatus to facilitate the immuno-cytochemistry process. Thus, for example, Shandon Scientific Limited manufacture the xe2x80x9cSequenzaxe2x80x9d(trademark) and xe2x80x9cCadenzaxe2x80x9d(copyright) immuno-staining apparatus. Use of the xe2x80x9cSequenzaxe2x80x9d(trademark) apparatus involves attaching a coverplate to the slide to be processed, thereby forming a narrow channel between the slide and coverplate. The coverplate comprises a funnel-like open-ended portion which, when attached to a slide, forms an open-ended chamber, into which staining reagents are manually introduced. The reagent then flows, under the influence of gravity, into the channel between the slide and the attached coverplate. When a new reagent is introduced into the well it displaces the previous reagent into a waste collection tray at the bottom of the apparatus.
The xe2x80x9cCadenzaxe2x80x9d(copyright) apparatus utilises the same coverplate system but additionally provides programmable automated control means for applying reagents to the open-ended well of the assembled slide/coverplate.
In relation to processing of samples supported on membranes, such samples are commonly preparations of proteins (termed Western blots) or DNA (Southern blots) or RNA (Northern blots). The molecular species of interest is visualised on the supporting membrane using specific antibodies and an appropriate detection systems (in the case of Western blots) or by hybridisation with a labelled probe (in the case of Southern and Northern blots). These processing techniques currently require considerable manual processing of the membranes.
The prior art apparatus for processing samples on solid supports is therefore very simple and limited in the functions which it can perform. The present invention aims to provide apparatus capable of more general application.
In a first aspect the invention provides a support retaining member for use in processing a sample supported on a support, the member being such that when assembled together with a support it forms a support cell, the support cell comprising a substantially sealed chamber, the chamber being provided with a fluid inlet and a fluid outlet for the introduction and removal respectively of fluids used in processing the sample.
In a particular embodiment, part of the internal surface of the substantially sealed chamber is defined by the sample bearing surface of the support, which arrangement has the advantage of minimising the number of components involved.
Conveniently the support is a slide, or a membrane of the type known to those skilled in the art.
Where the support is a slide, the support retaining member will typically be of such dimensions that it may be used with slides of conventional size, (i.e. slides which typically are about 25.4 mm by 76.2 mm). Where the support is a membrane, the dimensions of the retaining member will similarly be of such dimensions that it may be used with membranes of conventional size (typically 80 mm by 120 mm), although membranes are rather more variable in size than slides.
In some embodiments the support retaining member comprises two opposed portions, the support being positioned between the opposed portions of the support retaining member when the support cell is assembled. Preferably at least that portion of the support retaining member which comes into contact with the fluids used to process the sample on the support is made of an inert material, such as a synthetic moulded plastics material. Perspex is found particularly suitable. The remainder of the support retaining member may generally be of the same or other material. e.g. glass, perspex or metal (such as duraluminium).
The components of the support cell are typically held together by clamping means. In certain embodiments the clamping means is capable of clamping together the portions of a plurality of support cells. Typically, from one to around twelve or sixteen support cells may be clamped simultaneously by a single clamping means. The support cells may preferably be arranged in the clamping means in a substantially horizontal or substantially vertical manner, although any position intermediate between these two positions may be possible. The support cells may, for example, be arranged side-by-side, or one in front of the other.
As an alternative, or in addition, to clamping means the support cell may be provided with biasing means, which biasing means tends to urge together the components of the support cell. Conveniently the biasing means will comprise one or more sprung biasing members. In a particular embodiment, the support retaining member is attached to clamping means by spring-loaded mounting pins, such that formation of the support cell places the springs of the spring-loaded mounting pins under compression, which springs therefore urge together the components of the support cell.
In preferred embodiments, the force applied to the support cell by the clamping means and/or the biasing means helps to ensure a fluid-tight seal between the support and the support retaining member.
It is generally preferred that the support cell additionally comprises sealing means to assist in the formation of the substantially sealed chamber. Conveniently the sealing means forms a fluid-tight seal, in the first instance with the sample bearing surface of the support and, in the second instance, with the support retaining member. Alternatively, the sealing means may effect a seal directly between opposed portions of the support retaining member, where the support retaining member is of two-part construction. The sealing means may be an integral part of the support retaining member, or may be provided as a separate component of the support cell. The sealing means typically comprises a gasket, which may be made of silicon rubber or other suitable material. In one embodiment the sealing means comprises an O-ring gasket, the shape of which is generally that of a frame-like surround seated in a groove in one portion of the support retaining member. In an alternative embodiment the sealing means comprises a flattened frame-like surround gasket (about 100 to 150 xcexcm thick). Either type of gasket may be discarded after a single use (if, for example, contaminated with a radioactive probe) or may be re-used if desired. The flattened gasket embodiment is particularly suitable as a disposable gasket, to be discarded after a single use. It will be apparent that the thickness of the gasket (which can be readily altered by exchanging gaskets) may, in part, determine the volume of the substantially sealed chamber.
Conveniently the fluid inlet into, and the fluid outlet out of, the substantially sealed chamber are formed in the support retaining member. Where the support is a slide, the fluid inlet and fluid outlet are preferably provided in one portion where the support retaining member is of the two-part construction defined above. Where the support is a membrane, however, it is preferred that the fluid inlet and fluid outlet are provided in opposed portions of the support retaining member, where the support retaining member is of two-part construction. This encourages correct flow of processing fluids through the membrane.
The fluid inlet allows the introduction into the substantially sealed chamber of fluids needed to process the sample on the support. Typically such fluids will be buffers, solvents (e.g. ethanol/methanol, xylene), reagents (e.g. antibody- or probe-containing solutions) or stains. The fluid outlet allows for the processing fluids to be removed from the sample (e.g. for a washing step, or to allow the addition of a further reagent). Preferably, when the supports are being processed, their orientation is such that the fluid inlet is in the bottom portion of the substantially sealed chamber, and the fluid outlet is in the top portion of the substantially sealed chamber.
Typically, where the sample is supported on a slide, the substantially sealed chamber will have a volume of between 50 xcexcl and 300 xcexcl, preferably between 100-150 xcexcl. This small volume allows for economical use of reagents and (where temperature regulation is involved) a rapid thermal response time. Where the sample is supported on a membrane, the chamber will generally be larger (up to 2-3 mls).
In a particular embodiment, the support cell is adapted so as to be suitable for use in performing PCR on samples supported on a support (typically a slide), which hitherto has proved extremely difficult: performing PCR involves heating small volumes of liquids to comparatively high temperatures, which conventionally necessitates performing the PCR in a sealed reaction chamber to prevent undue evaporation loss of the reactants. However, the sealed chamber must be openable to allow the addition of further reagents. Such an openable chamber has hitherto been very difficult to create in the context of a standard slide. At present, the sealed chamber is typically created by placing a glass coverslip over a volume of PCR mix and sealing around the coverslip with a substance such as mineral oil or nail varnish. Re-opening the chamber is difficult and contamination of the chamber contents sometimes occurs. Also, the presence of the slide has hindered the ability rapidly to alter the temperature of the reagents, which ability is desirable when performing PCR.
In a particular embodiment envisaged for PCR (or other processes where temperature regulation is required), the support cell is equipped with temperature control means to allow for rapid heating and cooling of the sample and PCR mix (i.e. thermal cycling). Typically the support cell will be provided with an electrical heating element or a Peltier device. The support cell may also be adapted (e.g. by provision of cooling fins) to provide for improved air cooling. Temperature control in the range 4xc2x0-100xc2x0 C. is found sufficient for most applications.
In a further aspect, the invention provides automated apparatus for processing a sample, especially a biological sample, supported on a support, the apparatus comprising: support holding means for holding one or more supports, the sample on the or each support being present within a respective substantially sealed chamber; fluid delivery means for delivering processing fluid to the or each chamber; waste fluid collecting means for removing fluid from the or each chamber; and computer control means.
Preferably the apparatus is used in conjunction with the support cell defined above. Conveniently the holding means comprises clamping means suitable for clamping together the components of the support cell, as defined previously. Conveniently the holding means can accommodate from ten to twenty-four support cells.
A number of arrangements for appropriate fluid delivers means can be envisaged. In a preferred embodiment a number of reservoirs (typically, 10) of processing fluids, (e.g. buffers, stains, etc.) are provided, each reservoir being attached to pump means. Preferred pump means are syringe pumps, such as those manufactured by Hook and Tucker, (Croydon, Surrey, UK), or Kloen having a stroke volume of between 1 and 10 ml. One such pump may be provided for each processing fluid reservoir, or a single pump may be provided to pump fluid from each a plurality of reservoirs, by means of a multi-port valve configuration.
Conveniently each syringe pump is in turn attached to a central manifold (such as a universal connector). Preferably the central manifold feeds into a selective multi-outlet valve such that, if desired, where a plurality of samples are being processed simultaneously, each sample may be treated with a different processing fluid or combination of processing fluids, such that samples can be individually processed. A suitable selective multi-outlet valve is a rotary valve, such as the 10 outlet rotary valve supplied by Omnifit (Cambridge, UK). Thus each outlet from the multi-outlet valve may be connected to a separate support cell. One or more filters (especially xe2x80x9con-linexe2x80x9d filters) may be incorporated if desired. Typically a filter will be positioned between each reservoir and its associated syringe pump.
Each syringe pump may be actuated individually by the computer control means, or two or more pumps may be actuated simultaneously to provide a mixture of two or more processing fluids. Controlling the rate of operation of each pump will thus control the composition of the resulting mixture of processing fluids.
In an alternative embodiment, the fluid delivery means comprises two or more piston/HPLC-type pumps, each pump being supplied, via a multi-inlet valve, by a plurality of processing fluid reservoirs. Suitable pumps are available, for example, from Anachem (Luton, Beds, UK). Conveniently the multi-inlet valve will be a rotary valve. Each pump will feed into a rotary mixer, of the type well known to those skilled in the art, thus allowing variable composition mixtures of processing fluids to be produced, if desired.
Preferably, the processing fluid or mixture of processing fluids is then passed through an in-line filter and then conveniently passes through a selective multi-valve outlet (such as a rotary valve) before being fed into the support cells. Conveniently each support cell inlet will be provided with a respective valve.
As an alternative to the generally xe2x80x9cparallelxe2x80x9d supply of processing fluids defined above, the processing fluids may be supplied in xe2x80x9cseriesxe2x80x9d such that, for example, fluid is passed from one substantially sealed chamber to another. This embodiment would be particularly suitable, for example, when applied to simple staining of samples and has the advantage that the amount of reagent required is minimised.
In general relation to the fluid delivery means, it is a further preferred feature that a multi-inlet valve be located close to the fluid inlet provided in the or each substantially sealed chamber. Typically the valve will be a three-way valve with two inlets, and one outlet leading to the substantially sealed chamber. One of the valve inlets is fed, indirectly, by the reservoirs of processing fluid. The second inlet is conveniently-fed by a local reservoir which, typically, will be a syringe, pipette or micro-pipette (generally 100-5000 xcexcl volume). This local reservoir may be controlled by the computer control means or may be manually controlled. The local reservoir will typically be used where a reagent is scarce (e.g. an antibody) or where the reagent is radioactive (e.g. a radio-labelled probe or ligand). The provision of such a local reservoir minimises the amount of reagent required, simplifies radioactive decontamination procedures, and provides extra flexibility in that each support cell may be processed individually, if required.
Preferably processing fluid will enter the or each respective substantially sealed chamber at the bottom, travel upwards and exit from the chamber via the fluid outlet at the top. The or each fluid outlet will conveniently empty into a common collecting duct, which duct drains into a collecting vessel. The vessel is desirably removable from the apparatus to allow for periodic emptying or decontamination.
In a preferred embodiment, the apparatus is also equipped with temperature control means, as many procedures require some or all of the steps to take place at a specific temperature (e.g. binding of antibodies, hybridisation of probes and primers or labelled ligands). These temperature dependent steps are generally performed between 4xc2x0 C. and 95xc2x0 C. for between 30 minutes and 24 hours. The temperature control means may be incorporated into the support cells (e.g. in the form of heating elements). Alternatively or additionally the support holding means may comprise, or be formed with, or be situated within, temperature control means. In a particular embodiment the support holding means is mounted on a block provided with a Peltier device temperature control means.
Additionally, a fan may be provided to assist cooling of the apparatus.
The computer control means will generally comprise a personal computer (PC) or, more preferably, the computer control means will be integrated within the automated apparatus. Ideally, the computer control means should control two or more (preferably all) of the following parameters: the selection of which pump or pumps to actuate; the absolute volume and the rate of flow of processing fluid passing through the actuated pump(s); the selection of which support cell to feed with processing fluid; the temperature of the supported samples within the apparatus; and the timing of the various events.
It is envisaged that apparatus in accordance with the invention will be suitable for use, inter alia, in standard staining techniques, immuno- and enzyme cytochemistry (e.g. peroxidase/antiperoxidase staining); ligand binding studies; in situ hybridisation of labelled DNA or RNA probes; or even in situ PCR and in processing of Southern, Northern or Western blots. Preferably the computer control means has a programmable memory such that the steps needed to perform a certain procedure (e.g. haemotxylin/eosin staining) can be entered into the computer memory and readily re-called without the need to re-program the computer.
Conveniently the apparatus may be substantially modular such that, should large numbers of supported samples require to be processed, further reservoirs, pumps, support cells etc., can be readily added to the existing equipment. In such an embodiment the apparatus is preferably capable of accepting a modular array of support cells, whether the samples are supported on slides or membranes.
The invention further relates to manufacture of and use of the support cell and/or the apparatus of the invention in processing a sample on a support, such that the invention provides: a method of processing a sample on a support using a support cell and/or the automated apparatus defined above: a method of making a support cell and a method of making automated apparatus in accordance with the invention.